For many applications, it is desirable to be able to control gene expression at a particular stage in the growth of a plant or in a particular plant cell, tissue or part. For this purpose, methods are required which can provide for the desired initiation of transcription or expression in the appropriate cell types and/or at the appropriate time in a plant's development without having serious detrimental effects on plant development and productivity. In general, genetic engineering techniques have been directed to modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven more intransigent than other eukaryotic cells, due at least in part to a lack of suitable vector systems.
The geminiviruses are two-component single-stranded plant DNA viruses. They possess a circular single-stranded (ss) DNA as their genome encapsidated in twinned "geminate" icosahedral particles. The encapsidated ss DNAs are replicated through circular double stranded DNA intermediates in the nucleus of the host cell, presumably by a rolling circle mechanism. Viral DNA replication, which results in the simulation of both single and double stranded viral DNAs in large amounts, involves the expression of only a small number of viral proteins that are necessary either for the replication process itself or facilitates replication or viral transcription. The geminiviruses therefore appear to rely primarily on the machinery of the host to copy their genomes and express their genes.
Geminiviruses are subdivided on the basis of host range in either monocots or dicots and whether the insect vector is a leaf hopper or a white fly species. The molecular analysis of the genome of an increasing number of geminiviruses reinforces this division. All monocot-infecting geminiviruses are transmitted by leaf hoppers and their genome comprises a single ss DNA component about 2.7 kb in size; this type of genome, the smallest known infectious DNA, is typified by wheat dwarf virus which is one of a number from the subgroup that have been cloned and sequenced. By contrast, most members infecting dicot hosts are transmitted by the white fly Bemisia tabaci and possess a bipartite genome comprising similarly sized DNAs (usually termed A and B) as illustrated by African cassava mosaic virus (ACMV), tomato golden mosaic virus (TGMV) and potato yellow mosaic virus. For successful infection of plants, both genomic components are required. Beet curly top virus occupies a unique intermediary position between the above two subgroups as it infects dicots but contains only a single genomic component equivalent to DNA A possibly as a result of adaption to leaf hopper transmission.
The bipartite subgroup contains only the viruses that infect dicots. Exemplary is the African Cassava Mosaic Virus (ACMV) genome which comprises two circular single-stranded DNA molecules each of approximately 2.7 kb which contain a homologous region (approximately 200 nucleotides) known as the common region. From sequence and mutational analysis, DNA A is known to encode four open reading frames (ORFs). The ORFs are named according to genome component and orientation relative to the common region, i.e., complementary (c) versus viral (v): AC1, the polymerase gene essential to replication; AC2 is required for virus spread; AC3, is a regulator of DNA replication; and AVl is the coat protein gene. DNA B has two ORFs, BC1 and BV1, both of which are required for virus spread. The arrangement of the ORFs shows that they are expressed in a bidirectional manner. Five major transcripts have been identified and these map to the AV1, BV.sub.1, BC1, and ACI ORFs, separately and the AC2/AC3 ORFs together. AC2 has been shown to encode a transacting factor that stimulates production of the coat protein gene, AV1.
Another example from the bipartite group is the tomato golden mosaic virus (TGMV) which like ACMV is composed of two circular DNA molecules of the same size, both of which are required for infectivity. Sequence analysis of the two genome components reveals six open reading frames (ORFs); four of the ORFs are encoded by DNA A and two by DNA B. On both components, the ORFs diverge from a conserved 230 nucleotide intergenic region (common region) and are transcribed bidirectionally from double stranded replicative form DNA. The ORFs are named according to genome component and orientation relative to the common region (i.e., left versus right). The AL2 gene product transactivates expression of the TGMV coat protein gene, which is also sometimes known as "AR1".
There is little sequence analogy between the two DNA components of ACMV and TGMV, except for an almost identical common region of about 200 bases, however, the ORFs in the two genomes are analogous and there is the same requirement for the AL2 gene product, or the analogous AC2 gene product in ACMV, for transactivation of the coat protein gene. Inspection of AL2 sequences from several bipartite geminiviruses reveal that this protein has the general features expected of a transacting regulatory protein. It is possible that the requirement for AL2 function delays coat protein expression for a period of time sufficient to allow dsDNA amplification to occur.
Vectors in which the coat protein ORF has been replaced by a heterologous coding sequence have been developed and the heterologous coding sequence expressed from the coat protein promoter. However, since expression of the coat protein is dependent upon synthesis of the transacting regulatory protein, the timing of expression of the heterologous sequence from the coat protein promoter is dependent on the timing of expression of the transacting regulatory protein. Accordingly, it would be of interest to develop vectors in which the timing of the expression of the transacting regulatory protein is altered, thereby altering the timing of expression from the coat protein promoter and thus expression of a heterologous sequence inserted in place of the coat protein ORF.
The A genome component contains all viral information necessary for the replication and encapsidation of viral DNA, while the B component encodes functions required for movement of the virus through the infected plant. The DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B when inserted as a greater than full length copy into the genome of plant cells, or when a copy is electroporated into plant cells.